1. Field of the Invention
The invention relates to a reagent for detecting malaria infected cells and a detecting method for malaria infected cells using the reagent by which malaria infected cells, in particular malaria infected erythrocytes, can be stained rapidly and specifically.
2. Description of the Related Art
When a living body is infected by malaria or Plasmodium, malaria enters into erythrocytes and grows. Detecting malaria infected erythrocytes is an effective method for examining the possibility of the malaria infection.
Conventional methods for detecting malaria infected erythrocytes are as follows.
In one known method, malaria infected erythrocytes are detected by staining a blood smear with Giemsa stain and observing it with a microscope (see, for example, Toshio Nakamura et al., "An Examination Method for Plasmodium", Rinsho Kensa, Vol. 23, No. 4: 335-341, 1979). Another known method is staining a blood smear with a fluorescent dye and then observing with a fluorescent microscope to detect malaria infected erythrocytes (see Humihiko Kawamoto, "Simple and Rapid Staining for Diagnosis of Protozoal Infection with Fluorechromes", Rinsho Kensa, Vol. 32, No. 7: 803-806, 1988).
However, these methods are troublesome due to the necessity of steps for preparing, drying, fixing, and staining a blood smear, and also need a skilled technique for distinguishing malaria infected erythrocytes from erythrocytes which are not infected. Further, the microscopic observation takes a long time (generally more than 15 minutes).
A method for detecting malaria infected erythrocytes by using a flow cytometry has been also developed. In this method, malaria infected erythrocytes are stained with a fluorescent dye and detected by a flow cytometry. The mechanism of this method is that Plasmodium is stained with a fluorescent dye and fluoresces upon irradiating by an argon laser, whereby malaria infected erythrocytes are detected. Various methods using the above mechanism are known as follows.
(1) J. W. Jacobberger, "Analysis of Plasmodium-Infected Blood by Flow Cytometry", Cytometry, Vol. 4: 228-237, 1983
In this method, malaria infected erythrocytes are detected by a flow cytometry, using 3,3'-dimethyloxacarbocyanine: DiOCl (3) as a fluorescent dye. FIG. 7 of this document shows that the distribution of reticulocytes (Immature RBC: imRBC) and malaria infected erythrocytes (pRBC, ppRBC) are overlapped. Further, FIG. 2 is a photograph showing that the reticulocytes (imRBC) and malaria infected erythrocytes (pRBC, ppRBC) fluoresces by staining with DiOCl (3).
(2) M. T. Makler et al., "Thiazole Orange: A New Dye for Plasmodium Species Analysis", Cytometry, Vol. 8: 568-570, 1987
In this method, malaria infected erythrocytes are detected by a flow cytometry, using thiazole orange as a fluorescent dye. There is a description on page 569 in the middle to the left that reticulocytes are stained as a background staining.
(3) J. M. Whaum et al., "Rapid Identification and Detection of Parasitized Human Red Cells by Automated Flow Cytometry", Cytometry, Vol. 4: 117-122, 1983
In this method, malaria infected erythrocytes are detected by a flow cytometry, using acridine orange as a fluorescent dye. Since malaria infected erythrocytes contain malarial DNA, green fluorescence is emitted, while red fluorescence is emitted from reticulocytes owing to RNA contained in reticulocytes. Thus, reticulocytes can be distinguished from erythrocytes.
(4) K. Pattanapanyasat et al., "Flow Cytometric Two-Color Staining Technique for Simultaneous Determination of Human Erythrocyte Membrane Antigen and Intracellular Malarial DNA", Cytometry, Vol. 13: 182-187, 1992
This method uses propidium iodide (PI: emitting red fluorescence) as a fluorescent dye and an antigen to an antibody DAF (decay-accelerating factor) contained in erythrocytes membrane and measures an amount of erythrocyte membrane antigen and malaria infected erythrocyte.
The methods (1) and (2) described above have a drawback that malaria infected erythrocytes can not be accurately measured when reticulocytes and malaria infected erythrocytes are present together. The methods (3) and (4) require two measurements, i.e., red fluorescence and green fluorescence. Further, according to the method (3), since acridine orange which is used as a fluorescent dye, attaches to the passage of flow cytometry and measuring cell, significant contamination of the passage and the cell occurs. In the method (4), as monoclonal antibody is used, it takes more than 30 minutes to react with erythrocytes. Thus, these conventional methods have respective defects.
In addition to the references described above, followings are known.
(5) Japanese Laid-Open Patent Application 61-280565 (1986), corresponding to U.S. Pat. No. 4,985,174.
(6) Japanese Laid-Open Patent Application 62-034058 (1987).
These references disclose that a solution containing Auramine O as a fluorescent dye is mixed with a blood sample to stain reticulocytes with the fluorescent dye. As shown in the following table, four examples are described in the above reference (5), and one example in (6).
TABLE __________________________________________________________________________ (5) - 1 (5) - 2 (5) - 3 (5) - 4 (6) - 1 __________________________________________________________________________ Amount of Auramine O/ 1000 mg 400 mg 30 mg 400 mg 1000 mg 1 liter solution Solution: Blood (vol.) 5 ml: 5 ml: 5 ml: 2 ml: 10 ml: (Dilution ratio) 10 .mu.l 10 .mu.l 10 .mu.l 10 .mu.l 10 .mu.l Amount of Auramine O/ 500 .mu.g/ 200 .mu.g/ 15 .mu.g/ 80 .mu.g/ 1000 .mu.g/ 1 .mu.l blood .mu.l .mu.l .mu.l .mu.l .mu.l pH 7.2 8.0 9.0 8.0 7.2 Incubation temperature room room room room room temp. temp. temp. temp. temp. Incubation time 10 min. 10 min. 10 min. 30 sec. 30 sec. fluorescence wave 520 nm 580 nm 540 nm 520 nm 520 nm length for detecting or more or more or more or more or more __________________________________________________________________________
However, as described hereinafter, the reticulocytes and malaria infected erythrocytes are stained in the same level under the condition above. As a result, it is not possible to detect only the malaria infected erythrocytes.
Thus, conventional methods for detecting malaria infected erythrocytes retain defects to be improved.